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Toll-like receptor 6 gene polymorphisms increase the risk of bovine tuberculosis in Chinese Holstein cattle
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ARTICLE IN PRESS Acta Histochemica xxx (2014) xxx–xxx
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Toll-like receptor 6 gene polymorphisms increase the risk of bovine tuberculosis in Chinese Holstein cattle Yapan Song a,1 , Liping Sun b,c,1 , Aizhen Guo a , Liguo Yang c,∗ a b c
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China School of Food and Bioengineering, Zhengzhou University of Light Industry, Zhengzhou 450001, China China Education Ministry’s Key Laboratory in Agricultural Animal Genetics, Breeding and Reproduction, Wuhan 430070, China
a r t i c l e
i n f o
Article history: Received 10 February 2014 Received in revised form 7 June 2014 Accepted 9 June 2014 Available online xxx Keywords: TLR6 Bovine tuberculosis Polymorphism Chinese Holstein cattle
a b s t r a c t Our present study aimed to investigate the effect of four SNPs (G1793A, C1859A, A1980G, G1934A) in toll-like receptor 6 (TLR6) on bovine tuberculosis (bTB) resistance in a case–control study. A total of 603 Chinese Holstein cattle (264 from a dairy farm of Henan province, 339 from Hubei province) were selected to analyze the genotype of TLR6 gene by PCR-RFLP. Genotype frequencies of C1859A and A1980G site differed significantly between bTB-infected and non-infected cows (2 = 6.062, P = 0.048 and 2 = 6.749, P = 0.034, respectively). Relative risk of tuberculosis incidence result showed that genotypes of AA or CA had greater relative risk (OR = 2.730, 95%CI = 0.869–8.573; OR = 1.547, 95CI% = 0.803–2.982, respectively) than those with genotype CC at C1859A site between bTB-infected and non-infected animals. Genotypes of GG or GA had greater relative risk (OR = 2.986, 95%CI = 1.245–7.165; OR = 1.582, 95%CI = 0.734–3.409, respectively) than those with genotype AA at A1980G site. No significant association can be inferred from G1793A and G1934A polymorphism site. The present study suggests that variants in the TLR6 gene are associated with susceptibility to bTB and the TLR6 gene may be considered as a candidate gene for bTB resistance. © 2014 Published by Elsevier GmbH.
Introduction Bovine tuberculosis (bTB), as an endemic infectious disease, is mainly caused by Mycobacterium bovis (M. bovis). Bovine tuberculosis is still common in less developed countries and may cause severe economic losses such as deaths, chronic disease and trade restrictions (Proano-Perez et al., 2011). Studies have revealed that there are significant differences in the susceptibility to bovine tuberculosis between cattle breeds, for example, native Zebu cattle are more resistant to bovine tuberculosis than the exotic Holstein-Friesian cattle (Vordermeier et al., 2012). Genetic resistance to bTB was investigated and it was found that genetic heterozygosity, not only modulated resistance to bTB infection, but also influenced containment of disease progression in infected individuals and the role of genetic variability in natural populations was also confirmed (Acevedo-Whitehouse et al., 2005). Toll-like receptors (TLRs), by recognizing pathogen-associated molecular patterns (PAMPs), play important roles in innate
∗ Corresponding author. E-mail address: [email protected] (L. Yang). 1 These authors contributed equally to this work.
immune and adaptive immune responses (Zhang et al., 2009). The protein sequences of different TLRs within the same species or individual TLRs across different species are similar, however, their functions differ considerably (Kubarenko et al., 2007). Variants in TLR1, TLR2, as well as TLR4, 6 and 9 have been shown to be associated with susceptibility to pulmonary tuberculosis (PTB) in humans (Ma et al., 2010; Selvaraj et al., 2010). Researchers have found that TLR1, TLR2, TLR4, TLR6 and TLR9 play roles in the recognition of M. tuberculosis and components associated with the mycobacterial cell wall (Means et al., 1999; Bulut et al., 2001; Means et al., 2001; Bafica et al., 2005; Jo et al., 2007). TLR2 exerts its function often when heterodimerized with either TLR1 or TLR6 and can recognize mycobacterial antigens (Quesniaux et al., 2004). In our previous study, we investigated the effect of TLR1 gene polymorphisms on bovine tuberculosis susceptibility and found that TLR1 may be a candidate gene for bTB resistance (Sun et al., 2012). Researchers have confirmed that whole mycobacteria and many of their components act as ligands for TLRs (Means et al., 1999). To date, there are few reports devoted to investigating the role of TLR6 gene in bovine tuberculosis susceptibility, especially its gene polymorphisms. In order to illustrate the mechanism of TLR6 in bovine tuberculosis susceptibility, we examined the effect of TLR6 gene polymorphism on bTB susceptibility by PCR-RFLP.
http://dx.doi.org/10.1016/j.acthis.2014.06.004 0065-1281/© 2014 Published by Elsevier GmbH.
Please cite this article in press as: Song Y, et al. Toll-like receptor 6 gene polymorphisms increase the risk of bovine tuberculosis in Chinese Holstein cattle. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.06.004
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ARTICLE IN PRESS Y. Song et al. / Acta Histochemica xxx (2014) xxx–xxx
2
Materials and methods Samples and tuberculosis detection All procedures carried out on animals were approved by the Animal Care and Use Committee of Huazhong Agricultural University. The present study was conducted on 603 Chinese Holstein cows. The cows were 2–5 years of age; 264 were located in Henan province and 339 in Hubei Province. The climate of these two provinces was similar though the humidity of Hubei province may be somewhat higher than Henan province. The environmental and nutritional conditions were similar in these two dairy farms in order to control for an equal probability of bTB infection. Preliminary screening for TB was carried out using the purified protein derivative (PPD) tuberculin skin test (TST) according to the People’s Republic of China’s standards for diagnosing animal TB (GB/T 18645 2002). All of the procedures were conducted by one skilled veterinarian. Approximately 0.1 ml PPD was injected into the mid-neck, and the skin-fold thickness of each injection site was measured after 72 h. The reactions were then categorized as positive (>4 mm thickness with clinical signs such as diffuse or extensive edema, exudation, necrosis, pain or inflammation of the lymphatic ducts), negative (<2 mm thickness without clinical signs) or inconclusive (>2 mm thickness without clinical signs). Inconclusive cows were immediately re-tested on another section of the mid-neck using the same batch and dose of PPD. As the sensitivity of this test is less than 100%, another method, the Bovigam interferon gamma (IFN-␥) assay (Chen et al., 2008), was successfully used to confirm the status of the cows, especially those whose TST results were either inconclusive or negative. Cows were divided into infected (naturally infected) and non-infected (healthy) groups according to results of these two tests. Blood samples were drawn from the jugular veins into tubes containing 1.5% EDTA. Genomic DNA was isolated using the standard phenol–chloroform protocol, diluted to 50 ng/l according to its concentration and stored at −20 ◦ C for subsequent analysis.
were digested with BseLI, BstXI and BclI in a final volume of 10 L including 3 L of the PCR product, 5U of the restriction enzyme (NEB, Beijing, China), 1 L of the corresponding 10× reaction buffer and ultrapure water. The reaction was then incubated at 37 ◦ C for at least 4 h. Finally, the fragments were separated on a 3% agarose gel. Statistical analysis Data were analyzed using SAS 9.13 software (SAS Inst. Inc., Cary, NC, USA). Univariant analysis was performed for categorical variables with a 2 test on the distribution of allele and genotype frequencies between cases and controls. The relative risks of incidence for heterozygotes and for rare homozygotes relative to common homozygotes were estimated as odds ratios (ORs) with associated 95% confidence intervals (CIs). The relative risk of incidence among the genotypes was analyzed using a logistic regression model (log [p/(1 − p)] = ˛ + ˇ1 He + ˇ2 Va + Z; this model compared heterozygous (He) and homozygous (Va) genotypes to the common homozygous genotype and estimated two ORs, one for He and the other for Va, where ˛, ˇ1 , ˇ2 and were parameters) in 586 Chinese Holstein cattle (154 infected, 432 non-infected). ORs and 95% CIs were calculated according to Sole et al. (2006). Results and discussion Polymorphisms of the cattle TLR6genes Three genotypes AA (172 bp/45 bp), GA (172 bp/109 bp/63 bp/45 bp) and GG (109 bp/63 bp/45 bp) (Fig. 1a) were detected at the G1793A site. Three genotypes, AA (335 bp), CA (335 bp/178 bp/158 bp) and CC (178 bp/157 bp) were found at the C1859A site (Fig. 1b). For the small differences of 178 bp and 157 bp, we did not separate it clearly from the figure. A1980G site had three genotypes: GG (308 bp/27 bp), GA (308 bp/261 bp/47 bp/27 bp) and AA (261 bp/47 bp/27 bp) (Fig. 1c). Three genotypes were detected at the G1934A site: GG (508 bp/126 bp), GA (508 bp/197 bp/126 bp) and AA (508 bp/197 bp) (Fig. 1d).
Primers synthesis and genotyping Genotyping of the TLR6 gene polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Primers were designed using primer 5.0 software and synthesized by the Sangon Company (Table 1). The PCR reaction volume was 20 L, which included 10 L of PCR master mix, 0.5 L of each primer, 1 L of genomic DNA template, and8 L of ultrapure water. The PCR amplification conditions were optimized in a Gene Amp PCR System 9600 (Applied Biosystems, Foster City, CA, USA). The cycling program consisted of an initial denaturation (94 ◦ C for 5 min) followed by 35 cycles of 40 s at 94 ◦ C, 30 s at the annealing temperature, 30 s at 72 ◦ C, and a final extension of 7 min at 72 ◦ C. For RFLP, the PCR products
Effects of the polymorphisms on the relative risk of disease incidence To investigate the effects of different alleles and genotypes on bTB resistance, 2 tests and relative risks of incidence were analyzed in bTB-infected and non-infected cows. Studies have shown that variants in TLR6 gene were implicated in various inflammatory diseases (Hoffjan et al., 2005; Pierik et al., 2006; Misch et al., 2013). The results of our present study also showed that the genotype frequencies of the C1859A and A1980G polymorphisms differed significantly between bTB-infected and non-infected cows (2 = 6.062, P = 0.048 and 2 = 6.749 P = 0.034, respectively) (Table 2). This result does not conflict with previous
Table 1 Primers and amplification conditions. Site
Primer sequence
Annealing temperature (◦ C)
Product size (bp)
Product position
Restriction enzyme
G1793A
F: 5 -AGAACTCAACCTTGCTT-3 R: 5 -GGATACTTGGCCTACAC-3 F: 5 -GGTATCTTTAGCAGCCTTTCCATAC-3 R: 5 -GTCACAGCAACAGCCAGCA-3 F: 5’-ATTCCAATGTTCCTGTGAG-3’ R: 5’-CCGTGTTAATGTATTTCTGC-3’ F: 5’-GGTATCTTTAGCAGCCTTTCCATAC-3’ R: 5’-GTCACAGCAACAGCCAGCA-3’
50
217
CDS
BseLI
65
335
CDS
BstXI
50
798
CDS
BseLI
65
335
CDS
BclI
C1859A G1934A A1980G
Note: F: Forward; R: reverse; bp: base pairs; The GenBank numbers of TLR6 correspond to NM 001001159.
Please cite this article in press as: Song Y, et al. Toll-like receptor 6 gene polymorphisms increase the risk of bovine tuberculosis in Chinese Holstein cattle. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.06.004
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Fig. 1. Representative genotyping profile of the TLR6 gene at loci G1793A (a), C1859A (b), G1934A (c) and A1980G (d) by agarose gel electrophoresis. (a) Bands of 172 bp and 45 bp for AA genotype; 172, 109, 63 and 45 bp for GA genotype; 109, 63 and 45 bp for GG genotype. (b) Bands of 335 bp for AA genotype; 335, 178 and 157 bp for CA genotype; 178 bp and 157 bp for CC genotype, the small differences of 178 bp and 157 bp were not separated by agarose gel. (c) Bands of 508 and 126 bp for GG genotype; 508 bp and 197 bp for AA genotype; 508, 197 and 126 bp for GA genotype. (d) Bands of 308 bp and 27 bp for GG genotype; 308, 261, 47 and 27 bp for GA genotype; 261, 47 and 27 bp for AA genotype. The small fragments of 40 bp, 49 bp, 47 bp and 21 bp were invisible in the figure. M in a, b and d means 50 bp DNA Ladder, M in c means DL2000.
Table 2 2 and P-value of different genotypes between bTB-infected and non-infected cows. 2
P-value
227 (46.61) 162 (33.26) 98 (20.12)
0.062
0.969
6 (8.45) 54 (76.06) 11 (15.49)
19 (3.57) 381 (71.62) 132 (24.81)
6.062
0.048
AA GA GG
27 (48.21) 15 (26.79) 14 (11.11)
208 (47.93) 114 (26.27) 112 (88.89)
0.0185
0.991
AA GA GG
9 (16.07) 32 (57.14) 15 (26.79)
129 (26.27) 290 (59.06) 72 (14.66)
6.749
0.034
SNP
Genotype Cases
Controls
G1793A
AA GA GG
33 (45.83) 25 (34.72) 14 (19.44)
C1859A
AA CA CC
G1934A
A1980G
Genotype frequency
studies. Epigenetic DNA methylation in the promoters of the TLR1 and TLR6 gene is critically important for initial recognition of PAMPs (Al-Quraishy et al., 2013). Our previous studies have demonstrated that TLR1 gene polymorphisms play roles in bTB susceptibility (Sun et al., 2012). Statistically significant associations were also observed between variations in TLR2 and PTB (Velez et al., 2010). TLR6, also like TLR1, exerts its biological effects only when it forms a heterodimer with TLR2. Bovine tuberculosis has been on the increase in developed countries and continues to occur in developing countries (Gilbert et al., 2005). China, as a developing country, was also reported to have a high prevalence of bTB (Chen et al., 2009). High bTB prevalence was also detected in the two dairy herds of our study. The combination of these two assays increased the overall sensitivity and specificity of detection and minimized the incidence of false positives. In order to improve the reliability of our results, we used a large dairy herd to reduce the error between gene polymorphisms and disease susceptibility. TLRs play a crucial role in the immune recognition of Mycobacterium tuberculosis (M. tuberculosis). bTB as a contagious bacterial disease is caused by the bacteria M. tuberculosis. Interestingly, researchers have found that M. bovis and M. tuberculosis share a high degree of identity at the nucleotide (Garnier et al., 2003) and expression levels (Rehren et al., 2007) through in vitro and in vivo studies. Variants in TLR-1 (1805T/G), TLR-2 (2258G/A), TLR4 (896A/G and 1196C/T) and TLR-6 (745C/T) were investigated for their association with susceptibility or resistance to PTB (Ma et al., 2010; Selvaraj et al., 2010). In the present study, we investigated the effect of TLR6 gene polymorphisms (G1793A, C1859A, G1934A and A1980G) on susceptibility to bTB in a total of 603 Chinese Holstein
Table 3 Association of polymorphisms with relative risk of bTB incidence in cases (infected cows) and controls (healthy cows). SNP
Genotype
2
P-value
OR (95%CI)
G1859A
CC CA AA
6.062
0.048
1.000 1.547 (0.803–2.982) 2.730 (0.869–8.573)
A1980G
AA GA GG
6.749
0.034
1.000 1.582 (0.734–3.409) 2.986 (1.245–7.165)
cattle. Individual cows with AA or CA genotypes had a greater relative risk of bTB incidence [OR = 2.730, 95%CI (0.869–8.573); OR = 1.547, 95%CI (0.803–2.982), respectively] compared with cows with CC genotype at the C1859A site (Table 3). At the A1980G SNP, GG or GA genotype cows had a greater relative risk of bTB incidence [OR = 2.986, 95%CI (1.245–7.165); OR = 1.582, 95%CI (0.734–3.409), respectively] than AA genotype cows (Table 3). Although these two mutations do not change amino acid sequence, it has been observed that the synonymous SNP can affect in vivo protein folding and consequently function, as well as gene expression and phenotype (Kimchi-Sarfaty et al., 2007; Ren et al., 2010). This result of our study may illustrate that the genotype of TLR6 gene influences susceptibility to bTB. In conclusion, the results of the current study indicate that polymorphisms in the TLR6 gene are likely to be involved in susceptibility to bTB. These findings may contribute to the understanding of bTB pathogenesis and warrant further investigation to reveal the mechanisms of disease resistance in cattle.
Please cite this article in press as: Song Y, et al. Toll-like receptor 6 gene polymorphisms increase the risk of bovine tuberculosis in Chinese Holstein cattle. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.06.004
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Acknowledgements This study was supported by the earmarked fund for the Modern Agro-Industry Technology Research System of China (CARS-3704B), the Special Fund for Agro Scientific Research in the Public Interest (201003060), the Sino-UK Cooperation Key Program (S2010GR0947) and the National Basic Research Program of China (973 Program) (#2012CB518801). We thank the farm staff who helped to collect the data. References Acevedo-Whitehouse K, Vicente J, Gortazar C, Hofle U, Fernandezde-Mera IG, Amos W. Genetic resistance to bovine tuberculosis in the Iberian wild boar. Mol Ecol 2005;14:3209–17. Al-Quraishy S, Dkhil MA, Abdel-Baki AA, Delic D, Santourlidis S, Wunderlich F. Genome-wide screening identifies Plasmodium chabaudi-induced modifications of DNA methylation status of Tlr1 and Tlr6 gene promoters in liver, but not spleen, of female C57BL/6 mice. Parasitol Res 2013;112:3757–70. Bafica A, Scanga CA, Feng CG, Leifer C, Cheever A, Sher A. TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis. J Exp Med 2005;202:1715–24. Bulut Y, Faure E, Thomas L, Equils O, Arditi M. Cooperation of Toll-like receptor 2 and 6 for cellular activation by soluble tuberculosis factor and Borrelia burgdorferi outer surface protein A lipoprotein: role of Toll-interacting protein and IL-1 receptor signaling molecules in Toll-like receptor 2 signaling. J Immunol 2001;167:987–94. Chen Y, Chao Y, Deng Q, Liu T, Xiang J, Chen J, et al. Potential challenges to the Stop TB Plan for humans in China; cattle maintain M. bovis and M. tuberculosis. Tuberculosis 2009;89:95–100. Chen Y, Deng Q, Zhan Z, Guo A, Xiang J, Chen J, et al. Establishment of human IFN-gamma in vitro release assay and its application in tuberculosis diagnosis. Sheng wu gong cheng xue bao 2008;24:1653–7. Garnier T, Eiglmeier K, Camus JC, Medina N, Mansoor H, Pryor M, et al. The complete genome sequence of Mycobacterium bovis. Proc Natl Acad Sci USA 2003;100:7877–82. Gilbert M, Mitchell A, Bourn D, Mawdsley J, Clifton-Hadley R, Wint W. Cattle movements and bovine tuberculosis in Great Britain. Nature 2005;435:491–6. Hoffjan S, Stemmler S, Parwez Q, Petrasch-Parwez E, Arinir U, Rohde G, et al. Evaluation of the toll-like receptor 6 Ser249Pro polymorphism in patients with asthma, atopic dermatitis and chronic obstructive pulmonary disease. BMC Med Genet 2005; 6:34. Jo EK, Yang CS, Choi CH, Harding CV. Intracellular signalling cascades regulating innate immune responses to Mycobacteria: branching out from Toll-like receptors. Cell Microbiol 2007;9:1087–98. Kimchi-Sarfaty C, Oh JM, Kim IW, Sauna ZE, Calcagno AM, Ambudkar SV, et al. A “silent” polymorphism in the MDR1 gene changes substrate specificity. Science 2007;315:525–8.
Kubarenko A, Frank M, Weber AN. Structure–function relationships of Toll-like receptor domains through homology modelling and molecular dynamics. Biochem Soc Trans 2007;35:1515–8. Ma MJ, Xie LP, Wu SC, Tang F, Li H, Zhang ZS, et al. Toll-like receptors, tumor necrosis factor-alpha, and interleukin-10 gene polymorphisms in risk of pulmonary tuberculosis and disease severity. Hum Immunol 2010;71:1005–10. Means TK, Jones BW, Schromm AB, Shurtleff BA, Smith JA, Keane J, et al. Differential effects of a Toll-like receptor antagonist on Mycobacterium tuberculosis-induced macrophage responses. J Immunol 2001;166:4074–82. Means TK, Wang S, Lien E, Yoshimura A, Golenbock DT, Fenton MJ. Human toll-like receptors mediate cellular activation by Mycobacterium tuberculosis. J Immunol 1999;163:3920–7. Misch EA, Verbon A, Prins JM, Skerrett SJ, Hawn TR. A TLR6 polymorphism is associated with increased risk of Legionnaires’ disease. Genes Immun 2013;14:420–6. Pierik M, Joossens S, Van Steen K, Van Schuerbeek N, Vlietinck R, Rutgeerts P, et al. Toll-like receptor-1, -2, and -6 polymorphisms influence disease extension in inflammatory bowel diseases. Inflamm Bowel Dis 2006;12:1–8. Proano-Perez F, Benitez-Ortiz W, Portaels F, Rigouts L, Linden A. Situation of bovine tuberculosis in Ecuador. Revista panamericana de salud publica 2011;30:279–86. Quesniaux V, Fremond C, Jacobs M, Parida C, Nicolle D, Yeremeev V, et al. Toll-like receptor pathways in the immune responses to mycobacteria. Microbes Infect 2004;6:946–59. Rehren G, Walters S, Fontan P, Smith I, Zarraga AM. Differential gene expression between Mycobacterium bovis and Mycobacterium tuberculosis. Tuberculosis 2007;87:347–59. Ren G, Chen H, Zhang LZ, Lan XY, Wei TB, Li MJ, et al. A coding SNP of LHX4 gene is associated with body weight and body length in bovine. Mol Biol Rep 2010;37:417–22. Selvaraj P, Harishankar M, Singh B, Jawahar MS, Banurekha VV. Toll-like receptor and TIRAP gene polymorphisms in pulmonary tuberculosis patients of South India. Tuberculosis (Edinb) 2010;90:306–10. Sole X, Guino E, Valls J, Iniesta R, Moreno V. SNPStats: a web tool for the analysis of association studies. Bioinformatics 2006;22:1928–9. Sun L, Song Y, Riaz H, Yang H, Hua G, Guo A, et al. Polymorphisms in toll-like receptor 1 and 9 genes and their association with tuberculosis susceptibility in Chinese Holstein cattle. Vet Immunol Immunopathol 2012;147:195–201. Velez DR, Wejse C, Stryjewski ME, Abbate E, Hulme WF, Myers JL, et al. Variants in toll-like receptors 2 and 9 influence susceptibility to pulmonary tuberculosis in Caucasians, African-Americans, and West Africans. Hum Genet 2010;127:65–73. Vordermeier M, Ameni G, Berg S, Bishop R, Robertson BD, Aseffa A, et al. The influence of cattle breed on susceptibility to bovine tuberculosis in Ethiopia. Comp Immunol Microbiol Infect Dis 2012;35:227–32. Zhang YB, He FL, Fang M, Hua TF, Hu BD, Zhang ZH, et al. Increased expression of Toll-like receptors 4 and 9 in human lung cancer. Mol Biol Rep 2009;36:1475–81.
Please cite this article in press as: Song Y, et al. Toll-like receptor 6 gene polymorphisms increase the risk of bovine tuberculosis in Chinese Holstein cattle. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.06.004
ARTICLE IN PRESS Acta Histochemica xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Acta Histochemica journal homepage: www.elsevier.de/acthis
Toll-like receptor 6 gene polymorphisms increase the risk of bovine tuberculosis in Chinese Holstein cattle Yapan Song a,1 , Liping Sun b,c,1 , Aizhen Guo a , Liguo Yang c,∗ a b c
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China School of Food and Bioengineering, Zhengzhou University of Light Industry, Zhengzhou 450001, China China Education Ministry’s Key Laboratory in Agricultural Animal Genetics, Breeding and Reproduction, Wuhan 430070, China
a r t i c l e
i n f o
Article history: Received 10 February 2014 Received in revised form 7 June 2014 Accepted 9 June 2014 Available online xxx Keywords: TLR6 Bovine tuberculosis Polymorphism Chinese Holstein cattle
a b s t r a c t Our present study aimed to investigate the effect of four SNPs (G1793A, C1859A, A1980G, G1934A) in toll-like receptor 6 (TLR6) on bovine tuberculosis (bTB) resistance in a case–control study. A total of 603 Chinese Holstein cattle (264 from a dairy farm of Henan province, 339 from Hubei province) were selected to analyze the genotype of TLR6 gene by PCR-RFLP. Genotype frequencies of C1859A and A1980G site differed significantly between bTB-infected and non-infected cows (2 = 6.062, P = 0.048 and 2 = 6.749, P = 0.034, respectively). Relative risk of tuberculosis incidence result showed that genotypes of AA or CA had greater relative risk (OR = 2.730, 95%CI = 0.869–8.573; OR = 1.547, 95CI% = 0.803–2.982, respectively) than those with genotype CC at C1859A site between bTB-infected and non-infected animals. Genotypes of GG or GA had greater relative risk (OR = 2.986, 95%CI = 1.245–7.165; OR = 1.582, 95%CI = 0.734–3.409, respectively) than those with genotype AA at A1980G site. No significant association can be inferred from G1793A and G1934A polymorphism site. The present study suggests that variants in the TLR6 gene are associated with susceptibility to bTB and the TLR6 gene may be considered as a candidate gene for bTB resistance. © 2014 Published by Elsevier GmbH.
Introduction Bovine tuberculosis (bTB), as an endemic infectious disease, is mainly caused by Mycobacterium bovis (M. bovis). Bovine tuberculosis is still common in less developed countries and may cause severe economic losses such as deaths, chronic disease and trade restrictions (Proano-Perez et al., 2011). Studies have revealed that there are significant differences in the susceptibility to bovine tuberculosis between cattle breeds, for example, native Zebu cattle are more resistant to bovine tuberculosis than the exotic Holstein-Friesian cattle (Vordermeier et al., 2012). Genetic resistance to bTB was investigated and it was found that genetic heterozygosity, not only modulated resistance to bTB infection, but also influenced containment of disease progression in infected individuals and the role of genetic variability in natural populations was also confirmed (Acevedo-Whitehouse et al., 2005). Toll-like receptors (TLRs), by recognizing pathogen-associated molecular patterns (PAMPs), play important roles in innate
∗ Corresponding author. E-mail address: [email protected] (L. Yang). 1 These authors contributed equally to this work.
immune and adaptive immune responses (Zhang et al., 2009). The protein sequences of different TLRs within the same species or individual TLRs across different species are similar, however, their functions differ considerably (Kubarenko et al., 2007). Variants in TLR1, TLR2, as well as TLR4, 6 and 9 have been shown to be associated with susceptibility to pulmonary tuberculosis (PTB) in humans (Ma et al., 2010; Selvaraj et al., 2010). Researchers have found that TLR1, TLR2, TLR4, TLR6 and TLR9 play roles in the recognition of M. tuberculosis and components associated with the mycobacterial cell wall (Means et al., 1999; Bulut et al., 2001; Means et al., 2001; Bafica et al., 2005; Jo et al., 2007). TLR2 exerts its function often when heterodimerized with either TLR1 or TLR6 and can recognize mycobacterial antigens (Quesniaux et al., 2004). In our previous study, we investigated the effect of TLR1 gene polymorphisms on bovine tuberculosis susceptibility and found that TLR1 may be a candidate gene for bTB resistance (Sun et al., 2012). Researchers have confirmed that whole mycobacteria and many of their components act as ligands for TLRs (Means et al., 1999). To date, there are few reports devoted to investigating the role of TLR6 gene in bovine tuberculosis susceptibility, especially its gene polymorphisms. In order to illustrate the mechanism of TLR6 in bovine tuberculosis susceptibility, we examined the effect of TLR6 gene polymorphism on bTB susceptibility by PCR-RFLP.
http://dx.doi.org/10.1016/j.acthis.2014.06.004 0065-1281/© 2014 Published by Elsevier GmbH.
Please cite this article in press as: Song Y, et al. Toll-like receptor 6 gene polymorphisms increase the risk of bovine tuberculosis in Chinese Holstein cattle. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.06.004
G Model ACTHIS-50877; No. of Pages 4
ARTICLE IN PRESS Y. Song et al. / Acta Histochemica xxx (2014) xxx–xxx
2
Materials and methods Samples and tuberculosis detection All procedures carried out on animals were approved by the Animal Care and Use Committee of Huazhong Agricultural University. The present study was conducted on 603 Chinese Holstein cows. The cows were 2–5 years of age; 264 were located in Henan province and 339 in Hubei Province. The climate of these two provinces was similar though the humidity of Hubei province may be somewhat higher than Henan province. The environmental and nutritional conditions were similar in these two dairy farms in order to control for an equal probability of bTB infection. Preliminary screening for TB was carried out using the purified protein derivative (PPD) tuberculin skin test (TST) according to the People’s Republic of China’s standards for diagnosing animal TB (GB/T 18645 2002). All of the procedures were conducted by one skilled veterinarian. Approximately 0.1 ml PPD was injected into the mid-neck, and the skin-fold thickness of each injection site was measured after 72 h. The reactions were then categorized as positive (>4 mm thickness with clinical signs such as diffuse or extensive edema, exudation, necrosis, pain or inflammation of the lymphatic ducts), negative (<2 mm thickness without clinical signs) or inconclusive (>2 mm thickness without clinical signs). Inconclusive cows were immediately re-tested on another section of the mid-neck using the same batch and dose of PPD. As the sensitivity of this test is less than 100%, another method, the Bovigam interferon gamma (IFN-␥) assay (Chen et al., 2008), was successfully used to confirm the status of the cows, especially those whose TST results were either inconclusive or negative. Cows were divided into infected (naturally infected) and non-infected (healthy) groups according to results of these two tests. Blood samples were drawn from the jugular veins into tubes containing 1.5% EDTA. Genomic DNA was isolated using the standard phenol–chloroform protocol, diluted to 50 ng/l according to its concentration and stored at −20 ◦ C for subsequent analysis.
were digested with BseLI, BstXI and BclI in a final volume of 10 L including 3 L of the PCR product, 5U of the restriction enzyme (NEB, Beijing, China), 1 L of the corresponding 10× reaction buffer and ultrapure water. The reaction was then incubated at 37 ◦ C for at least 4 h. Finally, the fragments were separated on a 3% agarose gel. Statistical analysis Data were analyzed using SAS 9.13 software (SAS Inst. Inc., Cary, NC, USA). Univariant analysis was performed for categorical variables with a 2 test on the distribution of allele and genotype frequencies between cases and controls. The relative risks of incidence for heterozygotes and for rare homozygotes relative to common homozygotes were estimated as odds ratios (ORs) with associated 95% confidence intervals (CIs). The relative risk of incidence among the genotypes was analyzed using a logistic regression model (log [p/(1 − p)] = ˛ + ˇ1 He + ˇ2 Va + Z; this model compared heterozygous (He) and homozygous (Va) genotypes to the common homozygous genotype and estimated two ORs, one for He and the other for Va, where ˛, ˇ1 , ˇ2 and were parameters) in 586 Chinese Holstein cattle (154 infected, 432 non-infected). ORs and 95% CIs were calculated according to Sole et al. (2006). Results and discussion Polymorphisms of the cattle TLR6genes Three genotypes AA (172 bp/45 bp), GA (172 bp/109 bp/63 bp/45 bp) and GG (109 bp/63 bp/45 bp) (Fig. 1a) were detected at the G1793A site. Three genotypes, AA (335 bp), CA (335 bp/178 bp/158 bp) and CC (178 bp/157 bp) were found at the C1859A site (Fig. 1b). For the small differences of 178 bp and 157 bp, we did not separate it clearly from the figure. A1980G site had three genotypes: GG (308 bp/27 bp), GA (308 bp/261 bp/47 bp/27 bp) and AA (261 bp/47 bp/27 bp) (Fig. 1c). Three genotypes were detected at the G1934A site: GG (508 bp/126 bp), GA (508 bp/197 bp/126 bp) and AA (508 bp/197 bp) (Fig. 1d).
Primers synthesis and genotyping Genotyping of the TLR6 gene polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Primers were designed using primer 5.0 software and synthesized by the Sangon Company (Table 1). The PCR reaction volume was 20 L, which included 10 L of PCR master mix, 0.5 L of each primer, 1 L of genomic DNA template, and8 L of ultrapure water. The PCR amplification conditions were optimized in a Gene Amp PCR System 9600 (Applied Biosystems, Foster City, CA, USA). The cycling program consisted of an initial denaturation (94 ◦ C for 5 min) followed by 35 cycles of 40 s at 94 ◦ C, 30 s at the annealing temperature, 30 s at 72 ◦ C, and a final extension of 7 min at 72 ◦ C. For RFLP, the PCR products
Effects of the polymorphisms on the relative risk of disease incidence To investigate the effects of different alleles and genotypes on bTB resistance, 2 tests and relative risks of incidence were analyzed in bTB-infected and non-infected cows. Studies have shown that variants in TLR6 gene were implicated in various inflammatory diseases (Hoffjan et al., 2005; Pierik et al., 2006; Misch et al., 2013). The results of our present study also showed that the genotype frequencies of the C1859A and A1980G polymorphisms differed significantly between bTB-infected and non-infected cows (2 = 6.062, P = 0.048 and 2 = 6.749 P = 0.034, respectively) (Table 2). This result does not conflict with previous
Table 1 Primers and amplification conditions. Site
Primer sequence
Annealing temperature (◦ C)
Product size (bp)
Product position
Restriction enzyme
G1793A
F: 5 -AGAACTCAACCTTGCTT-3 R: 5 -GGATACTTGGCCTACAC-3 F: 5 -GGTATCTTTAGCAGCCTTTCCATAC-3 R: 5 -GTCACAGCAACAGCCAGCA-3 F: 5’-ATTCCAATGTTCCTGTGAG-3’ R: 5’-CCGTGTTAATGTATTTCTGC-3’ F: 5’-GGTATCTTTAGCAGCCTTTCCATAC-3’ R: 5’-GTCACAGCAACAGCCAGCA-3’
50
217
CDS
BseLI
65
335
CDS
BstXI
50
798
CDS
BseLI
65
335
CDS
BclI
C1859A G1934A A1980G
Note: F: Forward; R: reverse; bp: base pairs; The GenBank numbers of TLR6 correspond to NM 001001159.
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Fig. 1. Representative genotyping profile of the TLR6 gene at loci G1793A (a), C1859A (b), G1934A (c) and A1980G (d) by agarose gel electrophoresis. (a) Bands of 172 bp and 45 bp for AA genotype; 172, 109, 63 and 45 bp for GA genotype; 109, 63 and 45 bp for GG genotype. (b) Bands of 335 bp for AA genotype; 335, 178 and 157 bp for CA genotype; 178 bp and 157 bp for CC genotype, the small differences of 178 bp and 157 bp were not separated by agarose gel. (c) Bands of 508 and 126 bp for GG genotype; 508 bp and 197 bp for AA genotype; 508, 197 and 126 bp for GA genotype. (d) Bands of 308 bp and 27 bp for GG genotype; 308, 261, 47 and 27 bp for GA genotype; 261, 47 and 27 bp for AA genotype. The small fragments of 40 bp, 49 bp, 47 bp and 21 bp were invisible in the figure. M in a, b and d means 50 bp DNA Ladder, M in c means DL2000.
Table 2 2 and P-value of different genotypes between bTB-infected and non-infected cows. 2
P-value
227 (46.61) 162 (33.26) 98 (20.12)
0.062
0.969
6 (8.45) 54 (76.06) 11 (15.49)
19 (3.57) 381 (71.62) 132 (24.81)
6.062
0.048
AA GA GG
27 (48.21) 15 (26.79) 14 (11.11)
208 (47.93) 114 (26.27) 112 (88.89)
0.0185
0.991
AA GA GG
9 (16.07) 32 (57.14) 15 (26.79)
129 (26.27) 290 (59.06) 72 (14.66)
6.749
0.034
SNP
Genotype Cases
Controls
G1793A
AA GA GG
33 (45.83) 25 (34.72) 14 (19.44)
C1859A
AA CA CC
G1934A
A1980G
Genotype frequency
studies. Epigenetic DNA methylation in the promoters of the TLR1 and TLR6 gene is critically important for initial recognition of PAMPs (Al-Quraishy et al., 2013). Our previous studies have demonstrated that TLR1 gene polymorphisms play roles in bTB susceptibility (Sun et al., 2012). Statistically significant associations were also observed between variations in TLR2 and PTB (Velez et al., 2010). TLR6, also like TLR1, exerts its biological effects only when it forms a heterodimer with TLR2. Bovine tuberculosis has been on the increase in developed countries and continues to occur in developing countries (Gilbert et al., 2005). China, as a developing country, was also reported to have a high prevalence of bTB (Chen et al., 2009). High bTB prevalence was also detected in the two dairy herds of our study. The combination of these two assays increased the overall sensitivity and specificity of detection and minimized the incidence of false positives. In order to improve the reliability of our results, we used a large dairy herd to reduce the error between gene polymorphisms and disease susceptibility. TLRs play a crucial role in the immune recognition of Mycobacterium tuberculosis (M. tuberculosis). bTB as a contagious bacterial disease is caused by the bacteria M. tuberculosis. Interestingly, researchers have found that M. bovis and M. tuberculosis share a high degree of identity at the nucleotide (Garnier et al., 2003) and expression levels (Rehren et al., 2007) through in vitro and in vivo studies. Variants in TLR-1 (1805T/G), TLR-2 (2258G/A), TLR4 (896A/G and 1196C/T) and TLR-6 (745C/T) were investigated for their association with susceptibility or resistance to PTB (Ma et al., 2010; Selvaraj et al., 2010). In the present study, we investigated the effect of TLR6 gene polymorphisms (G1793A, C1859A, G1934A and A1980G) on susceptibility to bTB in a total of 603 Chinese Holstein
Table 3 Association of polymorphisms with relative risk of bTB incidence in cases (infected cows) and controls (healthy cows). SNP
Genotype
2
P-value
OR (95%CI)
G1859A
CC CA AA
6.062
0.048
1.000 1.547 (0.803–2.982) 2.730 (0.869–8.573)
A1980G
AA GA GG
6.749
0.034
1.000 1.582 (0.734–3.409) 2.986 (1.245–7.165)
cattle. Individual cows with AA or CA genotypes had a greater relative risk of bTB incidence [OR = 2.730, 95%CI (0.869–8.573); OR = 1.547, 95%CI (0.803–2.982), respectively] compared with cows with CC genotype at the C1859A site (Table 3). At the A1980G SNP, GG or GA genotype cows had a greater relative risk of bTB incidence [OR = 2.986, 95%CI (1.245–7.165); OR = 1.582, 95%CI (0.734–3.409), respectively] than AA genotype cows (Table 3). Although these two mutations do not change amino acid sequence, it has been observed that the synonymous SNP can affect in vivo protein folding and consequently function, as well as gene expression and phenotype (Kimchi-Sarfaty et al., 2007; Ren et al., 2010). This result of our study may illustrate that the genotype of TLR6 gene influences susceptibility to bTB. In conclusion, the results of the current study indicate that polymorphisms in the TLR6 gene are likely to be involved in susceptibility to bTB. These findings may contribute to the understanding of bTB pathogenesis and warrant further investigation to reveal the mechanisms of disease resistance in cattle.
Please cite this article in press as: Song Y, et al. Toll-like receptor 6 gene polymorphisms increase the risk of bovine tuberculosis in Chinese Holstein cattle. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.06.004
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Please cite this article in press as: Song Y, et al. Toll-like receptor 6 gene polymorphisms increase the risk of bovine tuberculosis in Chinese Holstein cattle. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.06.004